1. Field of the Invention
This invention relates to antibody directed enzyme prodrug therapy (ADEPT) using a non-naturally occuring mutant form of a host enzyme, especially a mutant form of ribonuclease.
2. Description of the Related Art
Targeting of drugs selectively to kill cancer cells in a patient has long been a problem for medical research. ADEPT is one approach to overcome the problem. ADEPT uses a tumour selective antibody conjugated to an enzyme. The conjugate is administered to the patient (usually intravenously), allowed to localise at the tumour site(s) and clear from the general circulation. Subsequently a prodrug is administered to the patient which is converted by the enzyme (localised at the tumour sites) into a cytotoxic drug which kills tumour cells. Since one molecule of enzyme can catalyse generation of many cytotoxic drug molecules an amplification effect is produced. Furthermore tumour cells not displaying the antigen recognised by the antibody (tumours usually display microheterogeneity) are also killed by enzymically amplified generation of the cytotoxic drug. A known system uses the procaryotic enzyme carboxypeptidase G2 (CPG2) as the enzyme component (see WO 88/07378). A drawback of systems using procaryotic enzymes is that the normal gut flora may contain procaryotic organisms capable of triggering non-selective cytotoxic drug generation.
A further problem with known systems is that repeated administration of the conjugate results in a host immune response rendering the therapy less effective. The antibody component is generally a mouse monoclonal which can be humanised using known techniques to reduce immunogenicity. However reduction of the immunogenicity of the enzyme component has proved more problematic. This is because the enzyme component must not be present naturally in the human host circulation otherwise premature conversion of prodrug to cytotoxic drug will occur and no selective toxicity to tumours will be observed. Akzo in WO90/02939 have proposed use of human enzymes for ADEPT with selectivity being maintained by choice of a human enzyme not normally present in the circulation such as lysozyme. Akzo have chosen human lysozyme as their enzyme and because of the nature of the substrate requirements [being an endoglycosidase it requires .beta..sub.1-4 linked polymers of N-acetylglucosamine (NAG-chitin) for cleavage] they are forced into producing prodrugs containing such functionalities. To prevent cell entry they further elaborate the oligomer with taurine residues--relying on the sulphonic acids to prevent cell entry and hence cytotoxicity--20 fold less--FIG. 13 in WO90/02939.
Use of a mammalian enzyme such as alkaline phosphatase (Senter et al: U.S. Pat. No. 4,975,278) or a human enzyme such as beta-glucuronidase (Behringwerke DE 42336237) or lysozyme (Akzo; WO 90/07929) for ADEPT has the advantage that such enzymes should have reduced, or lack, immunogenicity compared with non-mammalian enzymes. A disadvantage of using a mammalian or human enzyme is that it is present endogenously in patients and there will thus be the potential for turnover for prodrug to drug which is not due to the administered antibody-enzyme conjugate. This is likely to lead to enhanced toxicity with this type of ADEPT approach. Prodrugs for alkaline phosphatase are rapidly converted to drugs both in mice (Doyle, T. W. and Vyas, D. M., Cancer Treatment Reviews 17, 127-131, 1990) and in man (Hande et al. Clinical Pharmacology and Therapeutics 53, 233, 1993) in the absence of any administered conjugate due to the widespread distribution of endogenous alkaline phosphatase, thus confirming this is a critical problem for this enzyme. Human data on prodrugs for beta-glucuronidase or lysozyme are not available. Glucuronidase and lysozyme are present in the plasma and in other tissue sites. Akzo report lysozyme is present in milk, tears, saliva, spleen, leukocytes and monocytes. Behringwerke in DE423637 report activated marcophages, granulocytes and platelets secrete glucuronidase. Since these cells are widely distributed throughout the body this could lead to undesirable prodrug activation. Indeed Behringwerke have shown in mice that after administration of a Doxorubicin prodrug relatively high levels of free drug accumulate in the spleen which is a rich source of these cell (see table 3 in DE4236237).
Use of human enzymes in this ADEPT approach is limited by the fact that only enzymes with a predominant intracellular distribution can be used and the prodrugs that are used with them must be kept out of cells to minimise toxicity. This severely limits the number of options to produce an ADEPT system. Lysozyme although being a small enzyme has disadvantages for ADEPT. Lysozyme does not release the active drug but releases a derivative of unknown pharmacological activity. In the example given by Akzo, Dox-(GlcNAc).sub.1 or Dox-(GlcNAc).sub.5 is released rather than free Doxorubicin. Glucuronidase can release the active drug e.g. adriamycin from the glucuronide prodrug and anti-tumour activities have been reported (Bosslet, K et al Cancer Research 54, 2151-59, 1994). However, human glucuronidase is a high molecular weight enzyme (150-300 KDa) and consequently the resulting targeting conjugate is likely to be very large. This is likely to cause problems with penetration into tissues such as a tumour since it is well documented that smaller proteins penetrate more rapidly into solid tumours. In addition glucuronidase is glycosylated and this glycosylation leads to the rapid blood clearance of the antibody-glucuronidase conjugate used in ADEPT. The rapid blood clearance results in little conjugate localising to tumour xenografts. The combination of high molecular weight and rapid blood clearance is likely to lead to poor tumour localisation in patients. Thus glucuronidase is not an ideal enzyme for ADEPT.
The present invention is based on the discovery that a host enzyme (for example human ribonuclease, an enzyme naturally present in the general circulation) can be engineered such that it will recognise a prodrug for ADEPT therapy that is not significantly recognised by natural host enzyme. Since the engineered enzyme is highly similar in terms of amino acid composition to the native host enzyme it advantageously exhibits markedly reduced immunogenicity compared with bacterial enzymes such as CPG2. The engineered enzyme does not occur naturally and thus non-selective triggering of prodrug activation, by natural flora or human enzymes, is advantageously reduced. The approach has the additional advantages that it is applicable to a wide range of human or mammalian enzymes since it is not limited by the natural distribution of the enzyme and prodrugs can be employed that get into cells.
These problems have been addressed in part by International patent application WO 95/13095 (Wellcome Foundation) which was published after the earliest priority date of the present invention. This application proposed ADEPT using mutant mammalian enzymes to activate prodrugs which are not activated by the corresponding native enzyme but did not disclose the presently claimed invention.
It is very surprising that the replacement of a charged residue, one located at or close to the substrate binding or catalytic site of an enzyme, by a residue of opposite charge, produces a mutant enzyme with an intact catalytic centre, and this mutant enzyme differs from the native enzyme solely in possessing a related, complementary but charged inverted substate specificity requirement.
Furthermore the prodrug/drug combinations disclosed in Wellcome (based on methotrexate and melphalan) rely on blockage of active transport mechanisms to prevent cell penetration of the prodrug. This limits the range of prodrug/drug possibilities to those possessing such active transport mechanisms. In contrast the reversed polarity approach disclosed herein allows choice of charge properties of prodrugs (which may or may not also possess active transport properties) to block cell entry of the prodrug and thus enable application of the invention to a wider range prodrug/drug options.